Morphological and Phylogenetic Analyses Reveal Three New Species of Distoseptispora (Distoseptisporaceae, Distoseptisporales) from Yunnan, China

Three new species of Distoseptispora, viz. D. mengsongensis, D. nabanheensis, and D. sinensis, are described and illustrated from specimens collected on dead branches of unidentified plants in Yunnan Province, China. Phylogenetic analyses of LSU, ITS, and TEF1 sequence data, using maximum-likelihood (ML) and Bayesian inference (BI), reveal the taxonomic placement of D. mengsongensis, D. nabanheensis, and D. sinensis within Distoseptispora. Both morphological observations and molecular phylogenetic analyses supported D. mengsongensis, D. nabanheensis, and D. sinensis as three new taxa. To extend our knowledge of the diversity of Distoseptispora-like taxa, a list of recognized species of Distoseptispora with major morphological features, habitat, host, and locality is also provided.

Distoseptispora is one of the Sporidesmium-like genera with high morphological similarity to the Sporidesmium Link and Ellisembia Subram. Distoseptispora species with euor distoseptate conidia covering the criteria of Ellisembia and Sporidesmium. Accordingly, Distoseptispora species cannot be classified based on morphology alone, as phylogenetic analysis showed that these genera are not closely related [1,2,22]. Distoseptispora also appears similar in conidial ontogeny to Aquapteridospora Jiao Yang, K.D. Hyde & Maharachch., but the latter has terminal and intercalary conidiogenous cells with circular scars, and produces 3-euseptate conidia [23,24]. Additionally, Distoseptispora formed a sister clade to Aquapteridospora, and is well separated with high support in phylogenetic trees [24]. Distoseptispora and Aquapteridospora belonged to the order Distoseptisporales Z.L. Luo [24,25].
Distoseptispora species are primarily found as saprobes on submerged wood, dead branches, culms, or leaves in freshwater or terrestrial habitats except for D. caricis Crous and D. palmarum S.N. Zhang, K.D. Hyde & J.K. Liu occurring on the leaves of Carex sp. and rachis of Cocos nucifera [3,6,26]. Species of the genus decompose lignocellulose in wood [22,27,28], but their ecological functions, geographical distribution, alpha-taxonomy, and teleomorph relationships are poorly known. During our continuing survey (2007-2022) of saprophytic microfungi from the forest ecosystems of southwest China, several Sporidesmium-like taxa were isolated on dead branches of unidentified perennial dicotyledonous plants from terrestrial habitats in Yunnan Province, China. Using multi-gene loci of LSU, ITS, and TEF1 sequence data, the systematic placement of these isolates represented several Distoseptispora species. Based on morphological characteristics and multi-locus phylogenetic analysis, three new species of Distoseptispora, viz. D. mengsongensis, D. nabanheensis, and D. sinensis, are proposed and described in this paper.

Sample Collection, Isolation, and Morphological Examination
Samples of decomposing wood and bark were collected from the forest floor in Yunnan Province, China, and brought them back to the laboratory in Ziploc™ bags. Samples were treated following the methods described by Ma et al. [29]. Colonies on the surface of dead branches were examined and visually observed with a stereomicroscope (Motic SMZ-168, Xiamen, China) from low (7.5 times) to high (50 times) magnification. Fresh colonies were picked with sterile needles at a stereomicroscope magnification of 50 times, placed on a slide with a drop of lactic acid-phenol solution (lactic acid, phenol, glycerin, sterile water; 1:1:2:1, respectively), then placed under an Olympus BX 53 light microscope fitted with an Olympus DP 27 digital camera (Olympus Optical Co., Ltd., Tokyo, Japan) for microscopic morphological characterization. The tip of a sterile toothpick dipped in sterile water was used to pick conidia from the specimen; the conidia were then streaked on the surface of potato dextrose agar (PDA; 20% potato + 2% dextrose + 2% agar, w/v) and incubated at 25 • C overnight. Single-spore isolations were made on potato dextrose agar (PDA) following Goh [30]. Colony colors were assessed according to the charts of Rayner [31]. All fungal strains were stored in 10% sterilized glycerin at 4 • C for further studies. The studied specimens and cultures were deposited in the Herbarium of Jiangxi Agricultural University, Plant Pathology, Nanchang, China (HJAUP).

DNA Extraction, PCR Amplification, and Sequencing
Fungal hyphae (500 mg) were scraped from the surface of colonies growing on PDA plates, transferred to 2 mL safe-lock microtubes, and ground with liquid nitrogen. DNA was extracted using the Solarbio Fungal Genomic DNA Extraction Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer's instructions. Primer sets were used for the amplification of LSU and ITS, and TEF1: ITS5/ITS4 [32], 28S1-F/28S3-R [8], and EF1-983F/EF1-2218R [33]. The final volume of the PCR reaction was 25 µL, containing 1 µL of DNA template, 1 µL of each forward and reward primer, 12.5 µL of 2 × Power Taq PCR MasterMix, and 9.5 µL of double-distilled water (ddH 2 O). The PCR thermal cycling conditions of ITS and LSU were initialized at 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 55 • C for 50 s, elongation at 72 • C for 1 min, a final extension at 72 • C for 10 min, and finally kept at 4 • C. TEF1 was initialized at 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 52 • C for 30 s, elongation at 72 • C for 1 min, a final extension at 72 • C for 10 min, and finally kept at 4 • C. The PCR products were checked on 1% agarose gel electrophoresis stained with ethidium bromide. Purification and DNA sequencing were carried out at Beijing Tsingke Biotechnology Co., Ltd., Beijing, China.

Sequence Alignment and Phylogenetic Analyses
Sequences, including those obtained from GenBank (Table 1), were initially aligned using MAFFTv.7 [34] on the online server (http://maffTh.cbrc.jp/alignment/server/, accessed on 1 February 2023) and optimized manually when needed. The LSU, ITS, and TEF1 sequence data were concatenated by using Phylosuite software v1.2.1 [35], and absent sequence data in the alignments were treated with the question mark and "-" as missing data. The phylogenetic tree was constructed using Phylosuite software v1.2.1 [35] based on the combined data of LSU, ITS, and TEF1 sequence (Supplementary Materials). The concatenated aligned dataset was analyzed separately using maximum likelihood (ML) and Bayesian inference (BI). Maximum-likelihood phylogenies were inferred using IQ-TREE [36] under edge-linked partition model for 10,000 ultrafast [37] bootstraps. The final tree was selected among suboptimal trees from each run by comparing the likelihood scores using the TNe+I+G4 for ITS, TNe+R3 for LSU, and TN+F+I+G4 for TEF1 substitution model. Bayesian inference phylogenies were inferred using MrBayes 3.2.6 [38] under partition model (2 parallel runs, 2,000,000 generations), in which the initial 25% of sampled data were discarded by burning. The best-fit model was GTR+F+I+G4 for ITS+TEF1, and GTR+F+I+G4 for LSU. ModelFinder was used to select the best-fit partition model (edge-linked) using BIC criterion [39]. The trees were viewed in FigTree v. 1.4.4 (http: //tree.bio.ed.ac.uk/software/figtree, accessed on 1 February 2023), and further edited in Adobe Illustrator 2021. Sequences generated in this study were deposited in GenBank (Table 1).  Notes: The ex-type cultures are indicated using " T " after strain numbers; "-" stands for no sequence data in GenBank.

Molecular Phylogeny
The combined sequence alignment comprised 72 strains representing 64 species (Table 1)
Culture characteristics: Colony on PDA reached 81-86 mm diam. after 2 weeks in an incubator under dark conditions at 25 • C, irregularly rounded, surface velvety, with dense, brown mycelium, margin entire, dark brown to black; the reverse is black.
Culture characteristics: Colony on PDA reaching 64-69 mm diam. after 2 weeks in an incubator under dark conditions at 25 • C, irregularly rounded, surface velvety, with dense, gray mycelium, black at the entire margin; the reverse is dark brown to black.

Discussion
Sporidesmium-like taxa have undergone convergent evolution, and the morphological characteristics used to delimit Sporidesmium-like genera are shown to be insignificant in a phylogenetic context. Responding to the heterogeneity of Sporidesmium, the genus Distoseptispora was introduced by Su et al. [1] based on multi-locus phylogenies together with morphology. In recent years, the number of Distoseptispora species steadily increased and currently reached 63 species, including D. mengsongensis, D. nabanheensis, and D. sinensis. In the phylogenetic tree (Figure 1), some Distoseptispora species form sister clades, but they show different morphological characteristics, such as how D. mengsongensis and D. fasciculata are clustered, but the conidia of D. mengsongensis are obclavate, constricted at the septa, especially in proximal parts, sometimes with percurrent regeneration forming a secondary conidium from the conidial apex, with an average conidial length/width ratio of 14.54, while the conidia of D. fasciculata are subcylindrical to obclavate, with an average conidial length/width ratio of 8.44. Distoseptispora nabanheensis and D. clematidis have a close phylogenetic relationship, but D. nabanheensis has obclavate, slightly constricted at the septa, brown to dark brown conidia, with an average conidial length/width ratio of 15.13, while D. clematidis has oblong, obclavate, cylindrical or rostrate, brown with green tinge conidia, with an average conidial length/width ratio of 10.29. Distoseptispora sinensis, D. tectonigena, and D. tectonae have different morphology of conidiophores and conidia, and comparisons of nucleotides between D. tectonae (MFLUCC 12-0291 T ) and our isolate (HJAUP C2044) showed 10 and 9 (2%, including five gaps) nucleotide differences in ITS and LSU regions, respectively; D. tectonigena (MFLUCC 12-0292 T ) and our isolate (HJAUP C2044) showed 21 and 8 (4%, including seven gaps; 1.2%, including five gaps) nucleotide differences in ITS and LSU regions, respectively. Considering this scenario, additional molecular data and morphological characteristics are required for verification and expansion.
To date, all Distoseptispora species were identified by morphological and phylogenetic analyses, which led to a better evaluation of their phylogenetic relationships and taxonomic placements. However, studies conducted on Distoseptispora have no universally accepted standards with respect to barcode selection for phylogenetic analyses. For instance, Su et al. [1] established the genus Distoseptispora, but the initial species had only LSU sequences. Tibpromma et al. [10] [20] and Zhang et al. [21] introduced 10 Distoseptispora species using LSU, ITS, TEF1, and RPB2. The recent studies also indicated that the use of only LSU and ITS sequences might be problematic in resolving the phylogeny of Distoseptisporaceae, as RPB2 and/or TEF1 usually increased phylogenetic resolution significantly. In our study, we conducted phylogenetic analyses based on combined LSU, ITS, and TEF1 sequences, and obtained good phylogenetic support. Our three species, viz. D. mengsongensis, D. nabanheensis, and D. sinensis, are considerably distinct from all other described Distoseptispora species by morphological characteristics and multi-locus phylogenetic analysis, and thus we are convinced that the newly introduced species are new to science.
Studies conducted to date on Distoseptispora are mainly focused on their alpha-taxonomy, and most species of this genus are known from dead parts of plants as saprobic fungi in aquatic and terrestrial habitats [5,11,12,17,[19][20][21]43], except D. caricis and D. palmarum which are reported, respectively, on leaves of Carex sp. and rachis of Cocos nucifera on [3,6,26], whereas we have little attention on their roles in ecosystem function. They total 63 valid species (Tables 2 and 3), 44 of which are from freshwater, and 19 are from terrestrial habitats. Most Distoseptispora species are described based on their anamorph alone, and only two species, D. hyalina and D. licualae (Table 3), are reported as sexual morphs based on molecular DNA data, but the connection of teleomorph and anamorph has not been proved by pure culture or sequence data. The genus Distoseptispora has mainly been reported in China (41 species) and Thailand (22 species) [4], and only a small amount of published information is recorded in other regions (e.g., Hungary, Malaysia, Puerto Rico, Sierra Leone) [4,6,44]. Thus, it is unclear whether it has a close relationship with geographic regions, but we expect that large-scale surveys of Distoseptispora in aquatic and terrestrial habitats within different geographic regions, ecological environments, and climatic conditions are needed. This will contribute to a comprehensive knowledge of the species diversity of this genus, and further evaluate their phylogenetic relationships and taxonomic placements by molecular methods.